The concerns surrounding large-scale purification of proteins are an increasingly important issue for the biotechnology industry. Numerous disorders have been the subject of protein or enzyme replacement therapy including, dystrophic epidermolysis bullosa, and lysosomal storage disorders such as Gaucher disease, Fabry disease and Pompe disease. The large scale protein production required to supply patients must be cost sensitive, have production efficiency and yield high quality product. The process of protein purification is lengthy in time, burdensome as well as costly. These disadvantages greatly affect the cost of protein replacement therapy and pose a formidable challenge to healthcare in general.
Protein production is mainly performed in cells, i.e., mammalian, bacterial or fungal engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Cells expressing the protein of interest are cultured in a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Purification requires separation of the desired protein from the mixture of compounds fed to the cells as well as from cellular debris in order to purity sufficient amounts in high quality for use as a human therapeutic
Procedures for purification of proteins from cell debris are lengthy and complex. Multiple and repeated steps required to remove the protein of interest greatly compromises the final protein yield and quality. In many instances, the protein must be functional upon purification.
Recombinant proteins expressed in an intracellular compartment of a biological expression system are generally released from the expression system cells by mechanical disruption in cases where there is a cell wall. Such mechanical methods include homogenization, microfluidization, nitrogen burst, ultrasonic, and bead agitation methods. Other methods include the addition of enzymes to partially degrade cell wall components followed by osmotic agents to induce rupture and release of periplasm contents. These methods combining enzymatic digestion and chemical treatment are largely used for expressed proteins targeted to the periplasmic space in gram negative bacteria. Cells that have no cell wall may be disrupted by osmotic pressure without addition of enzymes, or complete by disruption of the cell membrane using detergents or organic solvents. Disruption methods may be used in combination for enhanced efficiency.
Most of the previous methods are suitable only for release of proteins from the periplasmic compartment, or result in complete disruption of the cell compartment. When the cell is completely disrupted, DNA may be released from subcellular compartments and cause formation of a highly viscous liquid. The DNA can be sheared or enzymatically degraded to reduce viscosity and enable handling the process fluid stream during larger scale productions. These steps are used successfully for production of pharmaceutical grade proteins; however, each process step increases the complexity, time and cost of manufacturing